1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives

ABSTRACT

Derivatives of 1,2,4-Triazolo-[3,4-b]-1,3,4-thiadiazoles according to formula (I), as set forth below: 
                         
processes for production thereof, pharmaceutical compositions containing the same and the use thereof in treatment of cancer.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to new1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives, their methods ofproduction and use in cancer treatment.

BACKGROUND OF THE INVENTION

Cancer still remains a fatal health problem even if progress intherapeutics has been noted. Nowadays, a constant increase in deathsfrom different types of cancer has been observed and it is estimatedthat in 2030 deaths will reach 12 million. Usually, anticancer therapyis considered as a challenge because of its high toxicity whichaccompanies it. Main side effects of anticancer drugs are nausea,vomiting, diarrhea, alopecia and infections (which are usually caused byleukopenia). Consequently, the development of new no or less toxicactive anticancer drugs is of the utmost importance and necessity.

Heterocyclic compounds that include thiazolic or thiadiazolic chemicalstructures have shown very important biologic activities such asantibacterial [a) Holla, B.S., Shivananda, M. K., Akberali, P. M.,Baliga, S., Safeer, S., Farmaco, 1996, 51, 785. b) Zhang, Z. Y., Sum, X.W., Chu, C. H., Zhao, L., J. Chim. Chem. Soc., 1997, 44, 535. c)Demirbas, N., Demirbas, A., Karaoglu, S. A., Celik, E., Arkivoc., 2005,1, 75.], anticancer [a) Ibrahim, D. A., Eur. J. Med. Chem., 2009, 44,2776 b) Al-Masoudi, N. A., Al-Soud, Y. A., Nucleos:Nucleot. NucleicAcids, 2008, 27, 1034. c) Chowrasia, D., Karthikeyan, C., Choure, L.,Sahabjada, Gupta, M., Arshad, Md., Trivedi, P., Arab. J. of Chem., 2013,doi:10.1016/j.arabjc.2013.08.026. d) Ilango, K., Valentina, P., Eur. J.Chem., 2010, 1, 50.], antiviral [a) Kritsanida, M., Mouroutsou, A.,Marakos, P., Pouli, N., Papakonstantinou-Garoufalias, S., Pannecouque,C., Witvrouw, M., Declercq, E., Farmaco, 2002, 57, 253. b) Invidiata, F.P., Simoni, D., Skintu, F., Pinna, N., Farmaco., 1996, 51, 659. c)Srivastava, V., Sen, S., Shekar, R., Indian J. Chem., 1994, 33B, 344.],anti-inflammatory [a) Chawla, G., Kumar, U., Bawa, S., Kumar, J., J.Enzyme Inhib. Med. Chem., 2012, 27, 658. b) Amir, M., Harish, K., Javed,S. A., Eur. J. Med. Chem., 2008, 43, 2056. c) Prasad, A. R., Ramlingam,T., Bhaskar Rao A., Diwan, P. B., Sattur., Indian J. Chem., 1986, 25B,566.], analgesic [a) Srivastava, V., Sen, S., Shekar, R., Indian J.Chem., 1994, 33B, 344. b) Chawla, G., Kumar, U., Bawa, S., Kumar, J., J.Enzyme Inhib. Med. Chem., 2012, 27, 658.], antifungal [a)Karabasanagouda, T., Adhikari, A. V., Suchethasettey, N., Eur. J. Med.Chem., 2007, 42, 521. b) Tiwari, N., Chaturvedi, B., Nizamuddin., Agric.Biol. Chem., 1988, 52, 1229.] and anthelmintic [α) El-Khawass, S.M.,Khalil, M. A., Hazzaa, A. A., Bassiouny, H. A., Loutfy, N. F., Farmaco.1989, 44, 703. 2. lmitiaz, M., Kumar, V., Indian J. Chem., 1992, 31B,673.]. In older studies, some amino- and diimino thiadiazole derivativeshave shown anticancer activity [a) Hill, D. L. Cancer Chemother.Pharmacol. 1980, 4, 215. b) Nelson, J. A., Rose, L. M., Benette, L.Cancer Res. 1977, 37, 182. c) Tsukamoto, K., Suno, M., Igarashi, K.,Kozai, Y., Sugino, Y., Cancer Res. 1975, 35, 2631].

SUMMARY OF THE INVENTION

The present invention provides new1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives and theirpharmaceutically acceptable salts, their methods of production andbiologic activity. The compounds of the present invention can be appliedin anticancer therapies due to their anticancer activity which isaccompanied by low acute toxicity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazolesof formula (I) or pharmaceutically acceptable salts thereof

wherein,

R₁ and R₂ are the same or different and are selected from the groupconsisting of C₁-C₅ alkyl, phenyl, methylphenyl,

R₃ is selected from the group consisting of CH₂R₈, CH₂CH₂R₈, CH═CHR₈,CH₂CH₂CH₂R₈, CH₂CH═CHR₈, CH═CHCH₂R₈, CH═CH—OR₈, CH₂-OR₈, CH₂CH₂—OR_(B),CH═CH—NHR₈, CH₂—NHR₈, CH₂CH₂—NHR₈, CH═CH—SR₈, CH₂—SR₈, CH₂CH₂—SR₈,

substituted or unsubstituted phenyl, benzyl, pyridyl, pyrimidinyl,triazinyl, triazinanyl, oxazinyl, oxazinanyl, cyclohexanyl,cyclohexenyl, cyclohexadienyl, pyranyl, oxathianyl, piperidinyl,cyclopentanyl, cyclopentenyl, cyclopentadienyl, pyrrolidinyl, pyrrolyl,furanyl, oxazolidinyl, pyrazolidinyl, thiophenyl, oxathiinyl,oxathiolyl, oxathiolanyl, wherein the substituent or substituents areselected from the group consisting of methyl F, Cl, Br, I, NO₂, CN, NH₂,OCH₂X, CH₂X, CX₃, CH₂CH₂X, OH wherein X is selected from the groupconsisting of H, F, Cl, Br, I,

R₄, R₅, R₆, R₇ are the same or different and are selected from the groupconsisting of H, F, Cl, Br, I, NO₂, CN, NH₂, OCH₃, OH, NHCH₂CH₃,N(CH₃)₂,

R₈ is selected from the group consisting of

substituted or unsubstituted phenyl, benzyl, pyridyl, pyrimidinyl,triazinyl, triazinanyl, oxazinyl, oxazinanyl, cyclohexanyl,cyclohexenyl, cyclohexadienyl, pyranyl, oxathianyl, piperidinyl,cyclopentanyl, cyclopentenyl, cyclopentadienyl, pyrrolidinyl, pyrrolyl,furanyl, oxazolidinyl, pyrazolidinyl, thiophenyl, oxathiinyl,oxathiolyl, oxathiolanyl, wherein the substituent or substituents areselected from the group consisting of methyl F, Cl, Br, I, NO₂, CN, NH₂,OCH₂X, CH₂X, CX₃, CH₂CH₂X, OH wherein X is selected from the groupconsisting of H, F, Cl, Br, I,

R₉ is selected from the group consisting of NHR₁₀, N R₁₁ R₁₂,

R₁₀ is selected from the group consisting of C₁-C₅alkyl, phenyl,

R₁₁ and R₁₂ are the same or different and they are C₁-C₅alkyl.

Preferably, R₁ and R₂ are the same or different and are selected fromthe group consisting of C₁-C₃ alkyl, phenyl, methylphenyl. Morepreferably, R₁ and R₂ are methyl.

Preferably, R₃ is selected from the group consisting of CH₂R₈, CH₂CH₂R₈,CH═CHR₈, CH₂CH₂CH₂R₈, CH₂CH═CHR₈, CH═CHCH₂R₈,

substituted or unsubstituted phenyl, benzyl, pyridyl, wherein thesubstituent or substituents are selected from the group consisting ofmethyl, F, Cl, Br, I, NO₂, CN, NH₂, OCH₂X, CH₂X, CX₃, CH₂CH₂X, OH,wherein X is selected from the group consisting of H, F, Cl, Br, I. Morepreferably, R₃ is selected from the group consisting of CH═CHR₈,CH₂CH₂CH₂R₈,

substituted or unsubstituted phenyl, pyridyl, wherein the substituent orsubstituents are selected from the group consisting of F, Cl, NO₂.

Preferably, R₄, R₅, R₆, R₇ are the same or different and they areselected from the group consisting of H, Cl, Br, I, NH₂, OCH₃. Morepreferably, R₄ and R₇ are H, R₅ and R₆ are OCH₃.

Preferably, R₈ is selected from the group consisting of

substituted or unsubstituted phenyl, benzyl, pyridyl, wherein thesubstituent or substituents are selected from the group consisting ofmethyl, F, Cl, Br, I, NO₂, CN, NH₂, OCH₂X, CH₂X, CX₃, CH₂CH₂X, OH,wherein X is selected from the group consisting of H, F, Cl, Br, I. Morepreferably, R₈ is selected from the group consisting of

substituted or unsubstituted phenyl, pyridyl, wherein the substituent orsubstituents are selected from the group consisting of F, Cl, NO₂.

Preferably, R₁₀ is selected from the group consisting of C₁-C₃ alkyl,phenyl. More preferably, R₁₀ is methyl.

Preferably, R₁₁ and R₁₂ are the same or different and they are C₁-C₃alkyl. More preferably, R₁₁ and R₁₂ are methyl.

The compounds of formula (I) contain at least one basic group andconsequently they can form pharmaceutically acceptable salts throughtreatment with a suitable acid. Suitable acids include pharmaceuticallyacceptable organic acids as well as pharmaceutically acceptableinorganic acids. Examples of pharmaceutically acceptable salts includechloride, bromide, sulphate, phosphate, nitrate, acetate, propionate,butyrate, maleate, tartarate, citrate, lactate, oxalate, succinate andbenzoate.

The compounds of formula (I) or their pharmaceutically acceptable saltscan be used in the treatment of different types of malignant neoplasms(solid tumors, lymphomas and leukemia). Preferably, they are used in thetreatment of ovarian cancer, breast cancer, prostate cancer, lung cancerand lymphomas. More preferably, they are used in the treatment ofovarian adenocarcinoma and small cell and non-small cell lung cancer.

The compounds of formula (I) or their pharmaceutically acceptable saltsshow powerful cytostatic and cytotoxic action and low acute and systemictoxicity.

The present invention provides also pharmaceutical compositions whichcomprise a compound of formula (I) or a pharmaceutically acceptable saltthereof. Such a pharmaceutical composition can be formulated so that itcan be administrated through the appropriate route such as oral,intranasal, topical or parenteral administration. For example, such apharmaceutical composition can be formulated into tablet, capsule,powder, solution, suspension, ointment or gel. Such a compositiongenerally contains apart from a compound of formula (I) or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablecarrier. Such a carrier may comprise excipients well known in the art,such as diluents, binders, fillers, disintegrants, lubricants, solvents,thickening agents, suspending agents, gelling agents, buffers orpreservatives. These compositions can be prepared following methodswhich are well known in the art.

The present invention further provides a process for the preparation ofcompounds of formula (I) or their pharmaceutically acceptable salts.

A general route of synthesis of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoleis presented below in Scheme 1.

Sulfonyl chlorides (V) can be synthesized from the reaction of aromaticesters (VI) with chlorosulfonic acid. Sulfonamides (IV) can be producedthough the reaction of sulfonyl chlorides (V) with amines. Hydrazides(III) can be prepared from sulfonamides (IV) with the treatment withaqueous hydrazine according to the methodology of Camoutsis and hiscolleagues (Ezabadi I R, Camoutsis C, Zoumpoulakis P, Geronikaki A,Soković M, Glamocilija J, Cirić A. Bioorg Med Chem. 2008,16(3):1150-61). Steps 4 and 5 can be performed according to the methodof Mathew and his colleagues (Mathew, V.; Keshavayya, J; Vaidya, V. P.;Giles, D. Eur. J. Med. Chem., 2007, 42, 521). Thus, hydrazides (III) canreact with carbon disulfide and hydrazine, producing the amino triazoles(II) via the intermediates potassium salts of dithiocarbamates. Finally,amino triazoles (II) can be converted into 3,6-disubstitued1,2,4-triazolo[3,4-b]-1,2,4 thiazoles (I) reacting with acids in thepresence of phosphorus oxychloride.

EXAMPLE 1 Synthesis of 2-(2(chlorosulfonyl)-4,5-di methoxyphenyl)methylacetate

In a flask containing 2-(3,4-dimethoxyphenyl)methyl acetate (3.7 g,17.65 mmol), CHCl₃ (35 ml) is added and then chlorosulfonic acid (5.28ml, 79.4 mmol) dropwise at 0° C. under an Ar atmosphere. The solutionobtains a dark purple color and is stirred at room temperature for 4 h.The reaction is quenched by the gradual addition of water (35 ml) at 0°C., followed by extractions with DCM (3×35 ml), drying the organic layerwith Na₂SO₄, condensation and column chromatography with 2: 1 elutionsolvent PS/EA. The product is collected as a white solid in 88% yield.

Synthesis of 2-(N,N-dimethylsulfamoyl)-4,5-dimethoxy-phenylacetylhydrazide

In an autoclave system containing2-(2-(chlorosulfonyl)-4,5-dimethoxyphenyl)methyl acetate (450mg,1.46mmol) dissolved in THF (1.8m1), a solution of dimethylamine wasadded in THF 2M (1.47 mL, 2.92 mmol) at 0° C. The reaction is stirred atroom temperature for 1 h and a pale yellow solid is observed. After thereaction is completed, the solution is decanted into a flask with therequired amount of DCM, concentrated on a rotary evaporator and theproduct is collected as a pale yellow solid in 100% yield.

Synthesis of5-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-amino-3-mercapto-1,2,4-triazole

In a cold solution of2-(N,N-dimethylosulfamoyl)-4,5-dimethoxy-phenylacetyl hydrazide (0.01mol), in absolute ethanol (150 mL), potassium hydroxide (0.015 mol) andcarbon disulfide (0.015 mol) are added. The reaction mixture is stirredat room temperature for 20h. During the reaction, the intermediatepotassium salt of dithiocarbamate precipitates. Subsequently, dry ether(150 mL) is added in order to complete the crystallization of the formedsalt, which is obtained by filtration and further dryed with dry ether.

The salt as suspension in 80% aqueous hydrazine (0.02 mol), is stirredwhile heated under reflux for 2 h. The reaction mixture is cooled,dissolved in cold water and neutralized with 10% hydrochloric acid. Theprecipitate is collected by filtration, washed with cold water, driedand recrystallized in methanol.

Yield: 56%, M.p. 222-223° C. (CH₃OH), ¹H NMR (500 MHz, DMSO-d6) δ 13.30(s, 1H), 7.15 (s, 1H), 6.92 (s, 1H), 5.46 (s, 2H), 4.20 (s, 2H), 3.71(s, 3H), 3.68 (s, 3H), 3.22 (s, 6H), ¹³C NMR (126 MHz, DMSO-d6) δ 165.8,151.8, 151.5, 147.1, 127.7, 126.3, 115.6, 112.7, 55.9, 36.8, 28.1.Analysis: C₁₃H₁₉N₅O₄S₂ (373). Calc. %: C:41.82, H:5.09, N:18.76 Found:C:41.77, H:5.12, N:18.79

Synthesis of3-[2-(N,N-dimethylsulfamoyI)-4,5-dimethoxy-benzyl]-6-phenyl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

The mixture of5[-2-(N,N-dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-amino-3-mercapto-1,2,4-triazole(0.01 mol) and benzoic acid (0.01 mol) in dry phosphorous oxychloride(3.7 mL) is stirred while heated under reflux for 2 h. The reactionmixture is cooled to room temperature and then poured into ice. Theexcess of phosphorus oxychloride is neutralized with dry potassiumcarbonate and appropriate amount of potassium hydroxide until the pH ofthe reaction is above 8. The solid is filtered off, washed extensivelywith water and dried.

Yield: 74%, M.p. 212-214° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 7.95 (d, J=6.9Hz, 2H), 7.68 (m, 1 H), 7.63 (m, 2H), 7.30 (s, 1 H), 7.21 (s, 1 H), 4.78(s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.63 (s, 6H), I.R. v cm⁻¹ 1601(C═N), 1573, 1513, 1470, 1446 (C═C), 1265 (N—N═C), 1328 (S—O antisym.),1141 (S—O sym.), Analysis: C₂₀H₂₁N₅O₄S₂ (459). Calc. %: C:52.28, H:4.57,N:15.25 Found: C:52.25, H:4.53, N:15.27

EXAMPLE 2

According to the methodology of example 1, the synthesis of thefollowing thiazoles has been completed.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(4-chlorophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 52%. M.p. 228-229° C. (CH₃OH—CH₂Cl₂), ¹H-NMR (CDCl₃) δ 7.97 (d,J=8.5 Hz, 2H), 7.71 (d, J=8.6 Hz, 2H), 7.30 (s, 1H), 7.20 (s, 1H), 4.78(s, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.62 (s, 6H), I.R. v cm⁻¹1600(C═N), 1573, 1519, 1470 (C═C), 1272 (N—N═C), 1339(S—O antisym.),1138 (S—O sym), Analysis: C₂₀H₂₀N₅O₄S₂Cl (493.5). Calc. %: C:48.63,H:4.05, N:14.18 Found: C:48.65, H:4.01, N:14.21.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(2-chloro-4-nitrophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 65%. M.p. 216-217° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 8.57 (d, J=2.3Hz, 1H), 8.40 (dd, J=8.7, 2.3 Hz, 1 H), 8.27 (d, J=8.7 Hz, 1 H), 7.30(s, 1 H), 7.20 (s, 1 H), 4.80 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.61(s, 6H), I.R. v cm⁻¹ 1603(C═N), 1573, 1518, 1476 (C═C), 1269 (N—N═C),1331 (S—O antisym.), 1138 (S—O sym.), Analysis: C₂₀H₁₉N₆O₆S₂Cl (538.5).Calc. %: C:44.56, H:3.52, N:15.60. Found: C:44.53, H:3.55, N:15.63.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3-methyl-4-nitrophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 46%. M.p. 142-144° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 8.18 (d, J=8.5Hz, 1H), 8.08 (s, 1H), 8.02 (d, J=8.5 Hz, 1H), 7.30 (s, 1H), 7.22 (s,1H), 4.80 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.62 (s, 6H), I.R. v cm⁻¹1600(C═N), 1573, 1510, 1476 (C═C), 1271 (N—N═C), 1344 (S—O antisym.),1133 (S—O sym.), Analysis: C₂₁H₂₂N₆O₆S₂ (518). Calc. %: C:48.64, H:4.24,N:16.21. Found: C:48.68, H:4.21, N:16.25.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3,4,5-trimethoxyphenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 38%. M.p. 169-170° C. (CH₃OH),¹H-NMR (CDCl₃) δ 7.30 (s, 1H), 7.22(s, 1H), 7.14 (s, 2H), 4.78 (s, 2H), 3.89 (s, 6H), 3.84 (s, 3H), 3.80(s, 3H), 3.76 (s, 3H), 2.62 (s, 6H), I.R. v cm⁻¹ 1630(C═N), 1586, 1459,1414 (C═C), 1267 (N—N═C), 1333 (S—O antisym.), 1127 (S—O sym.),Analysis: C₂₃H₂₇N₅O₇S₂ (549). Calc. %: C:50.27, H:4.92, N:12.75. Found:C:50.25, H:4.96, N:12.78.

3-[2-(N,N-dimethylosulfamoyl)-4,5-dimethoxybenzyl]-6-(2-aminophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 36%. M.p. 219-220° C. (C₂H₅OH), ¹H-NMR (CDCl₃) δ 7.30 (s, 1H),7.23 (t, J=7.8 Hz, 1H), 7.20 (s, 1H),7.13 (m, 1H), 7.03 (d, J=8.35 Hz,1H), 6.82 (d, J=8.2 Hz, 1H), 4.75 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H),2.61 (s, 6H), I.R. v cm⁻¹ 2436, 3382, 3267 (N—H), 1608 (C═N), 1555,1514, 1474 (C═C), 1265 (N—N═C), 1328 (S—O antisym.), 1141 (S—O sym.),Analysis: C₂₀H₂₂N₆O₄S₂ (474). Calc. %: C:50.63, H:4.64, N:17.72. Found:C:50.60, H:4.61, N:17.75.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3-aminophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 20%. M.p. 164-165° C. (C₂H₅OH), ¹H-NMR (CDCl₃) δ 7.44 (dd, J=8.0,1.4 Hz, 1H), 7.29 (m, 1H), 7.28 (s, 1H), 7.16 (s, 1H), 6.93 (d, J=8.4Hz, 1H), 6.71 (bs, 2H, NH2), 6.68 (t, J=7.5 Hz, 1H), 4.81 (s, 2H), 3.84(s, 3H), 3.78 (s, 3H), 2.63 (s, 6H), I.R. v cm^(−1,) 3450, 3347 (N—H),1626 (C═N), 1579, 1512, 1473 (C═C), 1272 (N—N═C), 1322 (S—O antisym.),1129 (S—O sym.), Analysis: C₂₀H₂₆N₆O₄S₂ (474). Calc. %: C:50.63, H:4.64,N:17.72. Found: C:50.65, H:4.67, N:17.69.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(4-aminophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 18%. M.p. 179-181° C. (C₂H₅OH), ¹H-NMR (CDCl₃) δ 7.58 (dd, J=8.6,2.9 Hz, 2H), 7.29 (d, J=3.0 Hz, 1H), 7.19 (d, J=2.8 Hz, 1H), 6.68 (dd,J=8.6, 2.9 Hz, 2H), 6.17 (s, 2H), 4.72 (s, 2H), 3.84 (s, 3H), 3.78 (s,3H), 2.61 (s, 6H), I.R. v cm⁻¹ 3457, 3348, 3237 (N—H), 1603 (C═N), 1577,1518, 1461 (C═C), 1265 (N—N═C), 1331 (S—O antisym.), 1138 (S—O sym.),Analysis: C₂₀H₂₆N₆O₄S₂ (474). Calc. %: C:50.63, H:4.64, N:17.72. Found:C:50.60, H:4.59, N:17.70.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-benzyl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 18%. M.p. 178-179° C. (CH₃OH); ¹H-NMR (CDCl₃) δ 7.42-7.36 (m,4H), 7.36-7.30 (m, 1H), 7.28 (s, 1H), 7.13 (s, 1H), 4.69 (s, 2H), 4.44,(s, 2H), 3.84 (s, 3H), 3.76 (s, 3H), 2.57 (s, 6H), I.R. v cm⁻¹ 1601(C═N), 1565, 1517, 1475 (C═C), 1267 (N—N═C), 1339 (S—O antisym.), 1140(S—O sym.), Analysis: C₂₁H₂₃N₅O₄S₂ (473). Calc. %: C:53.27, H:4.86,N:14.80. Found: C:53.23, H:4.89, N:14.83.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3-methoxybenzyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 58%. M.p. 165-166° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 7.30 (d, J=8.0Hz, 1H), 7.29 (s, 1H), 7.13 (s, 1H), 6.97 (s, 1H), 6.94 (d, J=7.7 Hz,1H), 6.90 (dd, J=8.3, 2.5 Hz, 1H), 4.70 (s, 2H), 4.41 (s, 2H), 3.84 (s,3H), 3.77 (s, 3H), 3.75 (s, 3H), 2.57 (s, 6H), I.R. v cm⁻¹ 1607 (C═N),1581, 1515, 1491 (C═C), 1266 (N—N═C), 1334 (S—O antisym.), 1139 (S—Osym.), Analysis: C₂₂H₂₅N₅O₅S₂ (503). Calc. %: C:52.48, H:4.97, N:13.91.Found: C:52.44, H:4.95, N:12.88.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(4-methoxybenzyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 52%. M.p. 184-185° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 7.30 (d, J=8.6Hz, 2H), 7.29 (s, 1H), 7.13 (s, 1H), 6.94 (d, J=8.6 Hz, 2H), 4.69 (s,2H), 4.36 (s, 2H), 3.84 (s, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 2.57 (s,6H), I.R. v cm⁻¹ 1610 (C═N), 1571, 1514, 1476 (C═C), 1267 (N—N═C), 1340(S—O antisym.), 1140 (S—O sym.), Analysis: C₂₂H₂₅N₅O₅S₂ (503). Calc. %:C:52.48, H:4.97, N:13.91. Found: C:52.45, H:4.99, N:13.94.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3,4-dimethoxybenzyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 50%. M.p. 139-140° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 7.29 (s, 1 H),7.13 (s, 1 H), 6.99 (s, 1 H), 6.95 (d, J=8.2 Hz, 1 H), 6.90 (dd, J=8.3,2.0 Hz, 1H), 4.70 (s, 2H), 4.35 (s, 2H), 3.84 (s, 3H), 3.77 (s, 3H),3.75 (s, 3H), 3.73 (s, 3H), 2.58 (s, 6H), I.R. v cm⁻¹ 1602 (C═N), 1555,1516, 1462 (C═C), 1266 (N—N═C), 1336 (S—O antisym.), 1139 (S—O sym.),Analysis: C₂₃H₂₇N₅O₆S₂ (533). Calc. %: C:51.78, H:5.06, N:13.13. Found%: 0:51.81, H:5.10, N:13.15.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3-phenylpropyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 14%. M.p. 128-129° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 7.30-7.26 (m,3H), 7.23-7.17 (m, 3H), 7.12 (s, 1H), 4.69 (s, 2H), 3.82 (s, 3H), 3.77(s, 3H), 3.04 (t, J=7.4 Hz, 2H), 2.68 (t, J=7.7 Hz, 2H), 2.59 (s, 6H),2.03 (p, J=7.6 Hz, 2H), I.R. v cm⁻¹ 1600 (C═N), 1574, 1516, 1478 (C═C),1267 (N—N═C), 1334 (S—O antisym.), 1139 (S—O sym.), Analysis:C₂₃H₂₇N₅O₄S₂ (501). Calc. %: C:55.09, H:5.38, N:13.97. Found %: C:55.11,H:5.41, N:14.01.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(2-pyridinyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 32%. M.p. 198-199° C. (CH₃OH), ¹H-NMR (CDCl₃) δ 8.76, (s, 1H),8.23-8.00 (m, 2H), 7.69 (s, 1H), 7.37-7.15 (m, 2H), 4.79 (s, 2H), 3.83(s, 3H), 3.78 (s, 3H), 2.62 (s, 6H), I.R. v cm⁻¹ 1599 (C═N), 1576, 1517,1456 (C═C), 1270 (N—N═C), 1335 (S—O antisym.), 1137 (S—O sym.),Analysis: C₁₉H₂₀N₆O₄S₂ (460). Calc. %: C:49.56, H:4.34, N:18.26. Found%: C:49.60, H:4.31, N:18.30.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(4-pyridinyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 37%. M.p. 231-232° C. (C₂H₅OH), ¹H-NMR (CDCl₃) δ 8.85 (d, J=5.0Hz, 2H), 7.91 (d, J=5.0 Hz, 2H), 7.30 (s, 1H), 7.21 (s, 1H), 4.80 (s,2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.63 (s, 6H), I.R. v cm⁻¹ 1600 (C═N),1561, 1507, 1473, 1411 (C═C), 1274 (N—N═C), 1337 (S—O antisym.), 1138(S—O sym.), Analysis: C₁₉H₂₀N₆O₄S₂ (460). Calc. %: C:49.56, H:4.34,N:18.26. Found %: C:49.58, H:4.37, N:18.23.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3-bromo-5-pyridinyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 41%. M.p. 227-228° C. (C₂H₅OH), ¹H-NMR (CDCl₃) δ 9.10 (d, J=2.0Hz, 1H), 8.98 (d, J=2.2 Hz, 1H), 8.58 (s, 1H), 7.30 (s, 1H), 7.22 (s,1H), 4.79 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.63 (s, 6H), I.R. v cm⁻¹1600 (C═N), 1573, 1516, 1481, 1440, 1412 (C═C), 1270 (N—N═C), 1338 (S—Oantisym.), 1140 (S—O sym.), Analysis: C₁₉H₁₉BrN₆O₄S₂ (539). Calc. %:C:42.30, H:3.52, N:15.58. Found %: C:42.32, H:3.49, N:15.56.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-cinnamyl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 44%. M.p. 191-193° C. (CH₃OH—CH₂Cl₂); ¹H-NMR (CDCl₃) δ 7.81 (d,J=7.1 Hz, 2H), 7.64 (d, J=16.4 Hz, 1H), 7.60 (d, J=16.3 Hz, 1H),7.52-7.40 (m, 3H), 7.30 (s, 1H), 7.16 (s, 1H), 4.73 (s, 2H), 3.84 (s,3H), 3.80 (s, 3H), 2.62 (s, 6H), I.R. v cm⁻¹ 1630 (CH═CH), 1600 (C═N),1575, 1576, 1475 (C═C), 1267 (N—N═C), 1332 (S—O antisym.), 1138 (S—Osym.), Analysis: C₂₂H₂₃N₅O₄S₂ (485). Calc. %: C:54.43, H:4.74, N:14.43.Found %: C:54.45, H:4.71, N:14.39.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(E)-4-fluorostyryl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 73%. M.p. 210-212° C. ¹H NMR (500 MHz, DMSO-d6) δ 7.89 (dd,J=8.6, 5.6 Hz, 2H), 7.65 (d, J=16.4 Hz, 1H), 7.58 (d, J=16.4 Hz, 1H),7.36-7.25 (m, 2H), 7.16 (s, 1H), 4.72 (s, 2H), 3.84 (s, 3H), 3.79 (s,3H), 2.60 (s, 6H). I.R. v cm⁻¹ 2926, 2853, 1736, 1631, 1601, 1513, 1494,1474, 1387, 1337, 1268, 1231, 1139, 1156, 1048.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(E)-4-chlorostyryl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 72%. M.p. 229-231° C. ¹H NMR (500 MHz, DMSO-d6) δ 7.84 (d, J=8.6Hz, 2H), 7.64 (s, 2H), 7.53 (d, J=8.6 Hz, 2H), 7.30 (s, 1H), 7.15 (s,1H), 4.72 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.61 (s, 6H). ¹³C NMR(126 MHz, DMSO-d6) δ 165.7, 151.9, 147.2, 146.3, 139.4, 134.8, 133.3,129.8, 129.0, 127.9, 126.1, 119.0, 115.5, 112.7, 55.9, 36.8, 27.6. I.R.v cm⁻¹ 2934, 2844, 1726, 1633, 1516, 1488, 1473, 1410, 1381, 1339, 1268,1224, 1139, 1175, 1087.

3-[2-(N,N-dimethylsulfamoyl)-4,5dimethoxybenzyl]-6-(E)-3-fluorostyryl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 52%. M.p. 212-215° C. ¹H NMR (500 MHz, DMSO-d6) δ 7.92-7.73 (m,2H), 7.68 (td, J=8.1, 5.8 Hz, 1H), 7.55 (td, J=8.6, 2.4 Hz, 1H), 7.30(s, 1H), 7.21 (s, 1H), 4.78 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.63(s, 6H). I.R. v cm⁻¹ 2929, 2844, 1589, 1516, 1474, 1387, 1338, 1270,1228, 1174, 1155, 1140, 1042

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-phenoxymethyl-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 41%. M.p. 178-179° C. (CH₃OH—CH₂Cl₂), ¹H-NMR (CDCl₃) δ 7.37-7.31(m, 2H), 7.29 (s, 1H), 7.13 (s, 1H), 7.09 (d, J=7.7, 1.0 Hz, 2H),7.06-7.01 (m, 1H), 5.55 (s, 2H), 4.72 (s, 2H), 3.84 (s, 3H), 3.78 (s,3H), 2.58 (s, 6H), I.R. v cm⁻¹ 1598 (C═N), 1572, 1516, 1479 (C═C), 1267(N—N═C), 1337 (S—O antisym.), 1139 (S—O sym.), Analysis: C₂₁H₂₃N₅O₅S₂(489). Calc. %: C:51.53, H:4.70, N:14.31. Found %: C:51.51, H:4.67,N:14.33.

p3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-[2-(2-methoxyphenyl)ethyl]-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 37%. M.p. 154-155° C. (CH₃OH—CH₂Cl₂), ¹H-NMR (CDCl₃) δ 7.29 (s,1H), 7.21 (m, 1H), 7.17 (d, J=7.5 Hz, 1H), 7.10 (s, 1H), 6.95 (d, J=8.2Hz, 1H), 6.84 (t, J=7.26 Hz, 1H), 4.67 (s, 2H), 3.84 (s, 3H), 3.77 (s,3H), 3.75 (s, 3H), 3.29 (m, 2H), 3.01 (t, J=7.5 Hz, 4H), 2,56 (s, 6H),I.R. v cm⁻¹ 1601 (C═N), 1569, 1519, 1477 (C═C), 1267 (N—N═C), 1336 (S—Oantisym.), 1139 (S—O sym.), Analysis: C₂₃H₂₇N₅O₅S₂ (517). Calc. %:C:53.38, H:5.22, N:13.53. Found %: C:53.41, H:5.19, N:13.56.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-[2-(4-methoxyphenyl)ethyl]-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 41%. M.p. 151-152° C. (CH₃OH—CH₂Cl₂), ¹H-NMR (CDCl₃) δ 7.29 (s,1H), 7.18 (d, J=8.3 Hz, 2H), 7.11 (s, 1 H), 6.83 (d, J=8.1 Hz, 2H), 4.67(s, 2H), 3.83 (s, 3H), 3.77 (s, 3H), 3.71 (s, 3H), 2.99 (t, J=7.6 Hz,2H), 2.55 (s, 6H), I.R. v cm⁻¹ 1601 (C═N), 1569, 1519, 1477 (C═C), 1267(N—N═C), 1336 (S—O antisym.), 1139 (S—O sym.), Analysis: C₂₃H₂₇N₅O₅S₂(517). Calc. %: C:53.38, H:5.22, N:13.53. Found %: C: 53.35, H:5.24,N:13.50.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(3-chlorophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 45%. M.p. 237-239° C., ¹H NMR (500 MHz, CDCl₃) δ 7.86 (s, 1H),7.74 (d, J=7.7 Hz, 1H), 7.56 (d, J=8.0 Hz, 1 H), 7.53-7.39 (m, 2H), 6.99(s, 1 H), 4.90 (s, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 2.72 (s, 6H). ¹³CNMR (126 MHz, CDCl₃) δ 164.9, 153.1, 152.5, 147.7, 146.8, 135.7, 132.7,130.9, 130.8, 127.9, 127.03, 127.0 125.3, 114.4, 113.4, 56.4, 56.2,36.9, 27.9. I.R. v cm⁻¹ 3088, 3056, 2955, 2927, 2852, 2616, 1598, 1573,1518, 1482, 1470, 1439, 1405, 187, 1339, 1270, 1228, 1175, 1139, 1101,1078, 1043.

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(4-nitrophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 53%. M.p. 223-225° C., ¹H NMR (500 MHz, CDCl₃) δ 8.39 (d, J=8.7Hz, 2H), 8.08 (d, J=8.7 Hz, 2H), 7.45 (s, 1H), 7.00 (s, 1H), 4.94 (s,2H), 3.93 (s, 3H), 3.88 (s, 3H), 2.73 (s, 6H), ¹³C NMR (126 MHz, CDCl₃)δ 163.9, 153.1, 152.5, 150.1, 147.9, 147.1, 134.8, 128.2, 127.5, 126.9,124.7, 114.5, 113.1, 113.1, 56.4, 37.1, 28.0, I.R. v cm⁻¹ 3108, 3004,2931, 2837, 1600, 1534, 1571, 1518, 1471, 1351, 1337, 1270, 1227, 1138,1049

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(4-fluorophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 66%. M.p. 220-223° C., ¹H NMR (500 MHz, DMSO-d6) δ 8.02 (dd,J=8.8, 5.2 Hz, 2H), 7.48 (t, J=8.8 Hz, 2H), 7.30 (s, 1H), 7.20 (s, 1H),4.78 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.62 (s, 6H), I.R. v cm⁻¹2933, 2842,1602, 1517, 1475, 187, 1337, 1268, 1241, 1225, 1139, 1042

3-[2-(N,N-dimethylsulfamoyl)-4,5-dimethoxybenzyl]-6-(2,4-dinitrophenyl)-1,2,4-triazolo[3,4-b]-1,3,4-thiazole

Yield: 29%. M.p. 162-164° C., ¹H NMR (500 MHz, DMSO-d6) δ 8.74 (s, 1H),8.69 (d, J=8.4 Hz, 1H), 8.50 (d, J=8.6 Hz, 1H), 7.29 (s, 1H), 7.13 (s,1H), 4.74 (s, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 2.58 (s, 6H), ¹³C NMR(126 MHz, DMSO-d6) δ 161.2, 153.8, 151.9, 150.5, 149.3, 147.3, 146.3,128.2, 127.7, 127.6, 126.9, 126.1, 124.3, 115.4, 112.8, 55.9, 55.8,36.7, 27.6, I.R. v cm⁻¹ 2917, 2850, 1729, 1555, 1514, 1457, 1346, 1308,1270, 1223, 1137, 1042

EXAMPLE 3

In vitro Study of Cytostatic-cytotoxic Activity Against Human Cancer andLeukemia Cell Lines

The present example illustrates the cytostatic-cytotoxic effects of thefollowing compounds of the present invention against the human ovariancancer cell lines UWB1,289 (with mutant BRCA1), UWB1,289+BRCA1, OVCAR-3,SCOV-3, the human breast cancer cell lines MCF −7 [expressing estrogenreceptors, insulin-like growth factor binding proteins (IGFBP) BP-2,BP-4; BP-5), oncogene WNT7B], T-47D (positive for the expression of allsteroid receptors and the oncogene WNT7B), the human cancer cell line ofhormone-resistant prostate cancer PC-3, the human acute T-leukemia cellline MOLT-4 (do not show expression of the mutant P53 oncogene), and thehuman lung carcinoma cell line A549 bearing a mutation in the KRASoncogene (p.G12S c.34G>A).

The R3 substituent of the compounds studied is defined in Table 1:

TABLE 1 {acute over ( )}Evωση R₃ {acute over ( )}Evωση R₃ TS167 C₆H₅—TS50 4-CH₃O—C₆H₄CH₂— TS63 4-Cl—C₆H₄— TS51 3,4-CH₃O—C₆H₃CH₂— TS572-NH₂—C₆H₄— TS56 C₆H₅—OCH₂— TS70 3-NH₂—C₆H₄— TS53 C₆H₅—CH═CH— TS714-NH₂—C₆H₄— TS54 2-CH₃O—C₆H₄CH₂CH₂— TS61 2-Cl-4-NO₂—C₆H₃— TS554-CH₃O—C₆H₄CH₂CH₂— TS60 2-CH₃-4-NO₂C₆H₃— TS52 C₆H₅CH₂CH₂CH₂— TS593,4,5-CH₃O—C₆H₂— TS66

TS62 C₆H₅CH₂— TS65

TS58 3-CH₃O—C₆H₄CH₂— TS67

TS22 3-Cl—C₆H₄— TS25 4-F—C₆H₄—CH═CH— TS29 3-F—C₆H₄— TS264-Cl—C₆H₄—CH═CH—

Specific Characteristics of Ovarian Cancer Cell Lines

SKOV-3 (SKOV-3) (ATCC HTB 77) and OVCAR-3 (ATCC® HTB-161™)

Human ovarian adenocarcinoma cells, which are grown easily in monolayerculture with epithelial-like morphology. SKOV-3 are resistant to tumornecrosis factor (TNF) and to other cytotoxic drugs such as cisplatin,and adriamycin, while OVCAR-3 are resistant to adriamycin, cisplatin andmelphalan. These cell lines express androgens, estrogens andprogesterone receptors. When SKOV-3 or OVCAR-3 cells are subcutaneouslyimplanted in nude mice, SCID mice, a moderately differentiated tumordevelops, and the resulting model resembles primary ovarianadenocarcinoma in humans.

UWB1.289 (ATCC CRL-2945)

Human ovarian adenocarcinoma cells, which are grown easily in monolayerculture, have morphology of epithelial cancer cells and do not expressestrogenic and progesterone receptors. Also, UWB1.289 cells have mutatedthe p53 tumor suppressor gene and positive Wilms' tumor protein (WT)expression, have no functional BRCA1 gene and are positive for theexpression of cytokeratin 7 (CK-7), calretinin and Wilms' tumor protein(WT). Finally they are sensitive to ionizing radiation.

UBWB1.289+BRCA1 (ATCC CRL-2946)

Human ovarian adenocarcinoma cells, derived from UBWB1,289, in which thepresence of the BRCA1 gene has been restored. They grow easily inculture, creating monolayers, have morphology of epithelial cancer cellsand do not express estrogenic and progesterone receptors. They are notsusceptible to ionizing radiation and radioimmune chemical agents (e.g.,alkylating agents). Also, UWB1.289 cells have mutated the p53 tumorsuppressor gene, and are positive for the expression of cytokeratin 7(CK-7), calretinin and Wilms' tumor protein (WT).

Cell Culture

All cancer cell lines are stored in a liquid nitrogen tank in 2.5 mlcryovials (Corning-Costar, Cambridge, Mass.). DMSO (dimethylsulfoxide)act as a cryoprotectant and is added to prevent the formation of icecrystals which may lyse the cells. The Cells were grown as monolayercultures in T-75 flasks (Corning-Costar, Cambridge, Mass.) andmaintained at 37° C. in 5% CO₂ incubator & 100% relative humidity. Thecultures medium used are McCoy's 5a Medium, supplemented with 10% fetalbovine serum (FBS, Gibco) and 1% antibiotics (100 IU/ml penicillin/100pg/ml streptomycin), sterilized by filtration (Corning-Costar filter,diameter 0.2 μm, RPMI-1640 and 50% RPMI-1640 (Catalog No 30-2001) plus50% Mammary Epithelial Growth Medium (MEGM), supplemented with 10% fetalbovine serum (FBS, Gibco) and 1% antibiotics (100 IU/ml penicillin/100μg/ml streptomycin). The passage is performed every 3-4 to days asfollows: Cells are washed with sterile PBS (Phosphate buffered saline):8 g/l NaCl, 0,2 g/l KCl, 1,15 g/l Na₂HPO4_(καl) 0,2 g/l KH₂PO4. pH:7,4),add 2-3 ml 0.05% trypsin (Gibco 1:250) and 0.02% EDTA to cover themonolayer and incubate the flask at 3TC for 5-10 minutes. Trypsin-EDTA,is used mainly to detach the cells from the flask and prepare a singlecell suspension. Finally, the number of cells can be determined bydirect counting using a Neubauer chamber and cell viability isdetermined by staining the cells with trypan blue.

In Vitro Study

In the experimental study the in vitro effect of the compound type (I)was evaluated against ovarian cancer cell lines UWB1.289, UWB1.289+BRCA1Kai SKOV-3.The cells were plated in 96-well plate (MTP) at a density of1×10⁴cells/m1 per well and maintained for 72 h at 3TC in a 5% CO₂incubator and grown as monolayers. The selection of the initial numberof cells was made according to the rate of proliferation of the cellline, in order the cells throughout the experiment are in an exponential(log) growth phase. After 24 hours, cells were treated with 1-100 μmol/lof the tested compounds for 48 h. Experiments were carried out usingtriplicate wells. The viability of cultured cells was estimated by an(3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)metabolic assay as described previously (Finlay G J, Wilson W R andBaguley B C: Comparison of in vitro activity of cytotoxic drugs towardshuman carcinoma and leukaemia cell lines. Eur J Cancer Clin Oncol 22:655-662, 1986; Alley M C, Scudiero D A, Monks A, Hursey M L, CzerwinskiM J, Fine D L, et al. Feasibility of drug screening with panels of humantumor cell lines using a microculture tetrazolium assay. Cancer Res1988; 48:589-601). MTT (Sigma, St Louis, Mo., USA) was dissolved in PBSin a concentration of 5 mg/ml, filter sterilized, and stored at 4° C.MTT (50 μl of stock solution) was added to each culture and incubatedfor 3 h at 3TC to allow metabolization. Formazan crystals weresolubilized by DMSO (100 μl). Absorbance of the converted dye wasmeasured at a wavelength of 540nm on ELISA reader (BioTek, Winooski,Vt., USA).

The mean concentrations of each compound that generated 50% or total(100%) growth inhibition (GI₅₀ and TGI, respectively) as well as thecompound concentrations that produced cytotoxicity against 50% of thecultured cells [(half maximal inhibitory concentration (IC₅₀)] werecalculated using the linear regression method. Using seven absorbancemeasurements [time 24 h (Ct24), control growth 72 h (Ct72), and testgrowth in the presence of drug at five concentration levels (Tt72x)],the percentage of growth was calculated at each level of the drugconcentrations. The percentage growth inhibition was calculatedaccording to National Cancer Institute (NCI) as:[(i Tt72x)−(Ct24)/(Ct72)−(Ct24)]×100 for concentrations for whichTt72x>Ct24 and [(Tt72x)−(Ct24)/Ct24]×100 for concentrations for whichTt72x<Ct24

GI50: The mean concentration that causes 50% inhibition of cell growth(Growth Inhibition 50%). The calculation of the value is based on theformula:(Tt72x)−(Ct24)/(Ct72)-(Ct24)×100=50

TGI: The mean concentration that causes 100% inhibition of cell growth(Total Growth Inhibition). The value is calculated according to theformula:(Tt72x)−(Ct24)/(Ct72)−(Ct24)×100=0

IC50: The mean concentration that kills 50% of the cells (InhibitionConcentration 50%). The value is calculated according to the formula:(Tt72x)−(Ct24)/(Ct24)×100=50

Results

The results were analyzed with Student's t-test. P<0.05 was consideredto be statistically significant.

The results of the in vitro study are presented in Table 2.

TABLE 2 GI50 TGI IC50 GI50 TGI IC50 GI50 TGI IC50 Comp. (μM) (μM) (μM)(μM) (μM) (μM) (μM) (μM) (μM) UWB1.289 + TS53 12 28 42 OVCAR-3 5 12 25SKOV-3 8 16 32 BRCA1 TS63 8 64 >100 3 32 >100 8 38 >100 TS62 8 64 >100 868 >100 12 56 >100 TS50 16 >100 >100 5 86 >100 6 >100 >100TS66 >100 >100 >100 24 >100 >100 20 >100 >100 TS167 76 >100 >10065 >100 >100 80 >100 >100 TS71 90 >100 >100 97 >100 >100 >100 >100 >100TS57 85 >100 >100 95 >100 >100 >100 >100 >100 TS22 16 >100 >10090 >100 >100 60 100 >100 TS25 5 12 36 24 68 94 18 52 80 TS29 10 64 >10074 >100 >100 45 84 >100 TS26 11 37 54 41 87 >100 38 72 >100 UWB1.289TS53 12 56 >100 MCF-7 15 45 >100 T-47D 8 37 >100 TS63 30 50 >100 2250 >100 27 55 >100 TS62 13 >100 >100 18 >100 >100 24 >100 >100 TS5030 >100 >100 40 >100 >100 28 >100 >100TS66 >100 >100 >100 >100 >100 >100 >100 >100 >100 TS167 56 >100 >10065 >100 >100 52 >100 >100TS71 >100 >100 >100 >100 >100 >100 >100 >100 >100TS57 >100 >100 >100 >100 >100 >100 >100 >100 >100 TS22 88 >100 >100 2490 >100 48 97 >100 TS22 16 >100 >100 90 >100 >100 60 100 >100 TS25 2 460 16 51 86 12 34 76 TS29 10 67 >100 66 >100 >100 48 86 >100 TS26 8 2172 25 67 >100 78 >100 >100 PC-3 TS53 12 31 85 MOLT-4 4 21 36 A-549 9 2458 TS63 20 45 >100 6 45 78 14 36 92 TS62 27 >100 >100 7 55 >100 1571 >100 TS50 44 >100 >100 10 >100 >100 35 >100 >100 TS66 >100 >100 >10085 >100 >100 >100 >100 >100 TS167 62 >100 >100 42 >100 >100 74 >100 >100TS71 >100 >100 >100 55 >100 >100 >100 >100 >100 TS57 >100 >100 >10094 >100 >100 >100 >100 >100 TS22 >100 >100 >100 72 >100 >100 45 95 >100TS25 8 24 69 16 29 78 11 56 87 TS29 85 >100 >100 61 >100 >100 30 71 >100TS26 19 47 86 32 81 >100 20 90 >100

The 12 derivatives of 1,2,4,triazolo-[3,4-b]-1,3,4- thiadiazole TS167,TS70, TS61, TS60, TS59, TS58, TS51, TS56, TS54, TS55, TS52, TS65 exhibita potent cytostatic (IG50<100 μM) than cytotoxic anticancer activity atthe concentration tested, with 1050 >100 μM in all 9 human cancer celllines.

EXAMPLE 4

In Vivo study of toxicity in C57BI/6 mice

In vivo Acute Toxicity

The acute toxicity of the compounds was assessed from lethality bytesting different concentrations, starting at 100mg/kg. The therapeuticdose of tested compound is defined as LD10 (lethal dose for 10% ofanimals). For intraperitoneal (i.p.) treatment, stock solutions of thetested compounds were prepared immediately before use. They weresuspended in corn oil in the desired concentration following initialdissolution in 10% dimethylsulfoxide (DMSO). C57BI/6 male and femalewere used for toxicity studies. Mice were kept under conditions ofconstant temperature and humidity in sterile cages with water and food.

The results from acute toxicity study are presented in Table 3.

TABLE 3 Compound LD50 (mg/kg) LD10 (mg/kg) TS53 375 >500 TS63 430 >500TS62 480 >500 TS25 345 >500 TS26 365 >500 TS50 >500 >500 TS66 >500 >500TS167 >500 >500 TS71 >500 >500 TS57 >500 >500

It is notable that all the compounds produced relatively very low acutetoxicity on C57BI/6 mice. All LD10s from the i.p. administration of thetested 1,2,4 triazolo-[3,4-b]-1,3,4 thiadiazole derivatives were over350 mg/kg whereas LD50s were not reached in any case. For thederivatives TS50, TS66, TS167, TS71, TS57, TS167, TS70, TS61, TS60,TS59, TS58, TS51, TS56, TS54, TS55, TS52 and TS65, acute toxicity wasnot demonstrated at the higher of the i.p. administrated dosage and LD10s and LD50 s were not reached (>500 mg/kg).

The invention claimed is:
 1. A compound of formula (I) or apharmaceutically acceptable salt thereof:

wherein R₁ and R₂ are the same or different and are selected from thegroup consisting of C₁-C₅ alkyl, phenyl, methylphenyl, R₃ is selectedfrom the group consisting of: CH₂R₈, CH₂CH₂R₈, CH═CHR₈, CH₂CH₂CH₂R₈,CH₂CH═CHR₈, CH═CHCH₂R₈, CH═CH—OR₈, CH₂—OR₈, CH₂CH₂—OR₈, CH═CH—NHR₈,CH₂—NHR₈, CH₂CH₂—NHR₈, CH═CH—SR₈, CH₂—SR₈, CH₂CH₂—SR₈,

substituted or unsubstituted phenyl, benzyl, pyridyl, pyrimidinyl,triazinyl, triazinanyl, oxazinyl, oxazinanyl, cycloexanyl, cycloexenyl,cycloexadienyl, pyranyl, oxathianyl, piperldinyl, cyclopentanyl,cyclopentenyl, cyclopentadienyl, pyrrolidinyl, pyrrolyl, furanyl,oxazolidinyl, pyrazolidinyl, thlophenyl, oxathiinyl, oxathiolyl,oxathiolanyl, wherein the substituent or substituents are selected fromthe group consisting of methyl, F, Cl, Br, I, NO₂, CN, NH₂, OCH₂X, CH₂X,CX₃, CH₂CH₂X, OH, wherein X is selected from the group consisting of H,F, Cl, Br, I, R₄, R₅, R₆, R₇ are the same or different and selected fromthe group consisting of: H, F, Cl, Br, I, NO₂, CN, NH₂, OCH₃, OH,NHCH₂CH₃, N(CH₃)₂, R₈ is selected from the group consisting of:

substituted or unsubstituted phenyl, benzyl, pyridyl, pyrimidinyl,triazinyl, triazinanyl, oxazinyl, oxazinanyl, cycioexanyl, cycloexenyl,cycloexadienyl, pyranyl, oxathianyl, piperidinyl, cyclopentanyl,cyclopentenyl, cyclopentadienyl, pyrrolidinyl, pyrrolyl, furanyl,oxazolidinyl, pyrazolidinyl, thiophenyl, oxathiinyl, oxathiolyl,oxathiolanyl, wherein the substituent or substituents are selected fromthe group consisting of: methyl, F, Cl, Br, I, NO₂, CN, NH₂, OCH₂X,CH₂X, CX₃, CH₂CH₂X, OH, wherein X is selected from the group consistingof H, F, Cl, Br, I, R₉ is selected from the group consisting of: NHR₁₀,NR₁₁R₁₂, R₁₀ is selected from the group consisting of: C₁-C₅ alkyl,phenyl, R₁₁ and R₁₂ are the same or different and are C₁-C₅ alkyl. 2.The compound according to claim 1, wherein R₁ and R₂ are the same ordifferent and are selected from the group consisting of: C₁-C₃ alkyl,phenyl, methyphenyl.
 3. The compound according to claim 1 wherein R₁ andR₂ are methyl.
 4. The compound according to claim 1, wherein R₃ isselected from the group consisting of: CH₂R₈, CH₂CH₂R₈, CH═CHR₈,CH₂CH₂CH₂R₈, CH₂CH═CHR₈, CH═CHCH₂R₈,

substituted or unsubstituted phenyl, benzyl, pyridyl, wherein thesubstituent or substituents are selected from the group consisting ofmethyl, F, Cl, Br, I, NO₂, CN, NH₂, OCH₂X, CH₂X, CX₃, CH₂CH₂X, OH,wherein X is selected from the group consisting of: H, F, Cl, Br, I. 5.The compound according to claim 1, wherein R₃ is selected from the groupconsisting of: CH═CHR₈, CH₂CH₂CH₂R₈,

substituted or unsubstituted phenyl, pyridyl, wherein the substituent orsubstituents are selected from the group consisting of: F, Cl, NO₂. 6.The compound according to claim 1, wherein R₄, R₅, R₆, R₇ are the sameor different and are selected from the group consisting of: H, Cl, Br,I, NH₂, OCH₃.
 7. The compound according to claim 1, wherein R₄ and R₇are H, R₅ and R₆ are OCH₃.
 8. The compound according to claim 1, whereinR₈ is selected from the group consisting of:

substituted or unsubstituted phenyl, benzyl, pyridyl, wherein thesubstituent or substituents are selected from the group consisting of:methyl, F, Cl, Br, I, NO₂, CN, NH₂, OCH₂X, CH₂X, CX₃, CH₂CH₂X, OH,wherein X is selected from the group consisting of: H, F, Cl, Br, I. 9.The compound according to claim 1, wherein R₈ is selected from the groupconsisting of:

substituted or unsubstituted phenyl, pyridyl, wherein the substituent orsubstituents are selected from the group consisting of: F, Cl, NO₂. 10.The compound according to claim 1, wherein R₁₀ is selected from thegroup consisting of: C₁-C₃ alkyl, phenyl.
 11. The compound according toclaim 1, wherein R₁₀ is methyl.
 12. The compound according to claim 1,wherein R₁₁ and R₁₂ are the same or different and are C₁-C₃ alkyl. 13.The compound according to claim 1, wherein R₁₁ and R₁₂ are methyl. 14.The pharmaceutical composition comprising a compound of formula (I)according to claim 1 or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier.
 15. The compound according to claim1 or a pharmaceutically acceptable salt thereof for use in the treatmentof one of ovarian cancer, breast cancer, prostate cancer, leukemia, orlung cancer.